MXPA06003570A - Method and device for culturing live cells by coupling a bioreactor receiver with a selection automation. - Google Patents

Abstract

The invention relates to a method for continuously, semi-continuously or discontinuously treating a substrate (24) consisting in placing said substrate in a bioreactor receiver (1) and in exposing it to the action of a living cell culture (C1) which makes it possible to carry out a reaction (R1) on the substrate (24) and to which a medium is periodically inoculated with the aid of living cells (C2) improving said reaction. Said living cells (C2) are obtainable by selection from a population of dynamic living cells carried out by an automatic selection device (2) which is supplied by the same substrate (24) as the bioreactor receiver (1) and is originally inoculated with the living cells (C1) contained in the bioreactor receiver (1) tank and an operating device.

Description

PROCEDURE AND DEVICE FOR THE CULTIVATION OF CELLS LIVE BY COUPLING A BIOREACTOR CONTAINER WITH A AUTOMATIC SELECTOR The present invention relates to a method and a device for the cultivation of living cells by coupling a bioreactor vessel with an automatic selector. Waste treatment is an increasingly constant concern of citizens and governments. An important part of the treatments is carried out in factories that put into practice tanks inoculated with a bacterial flora. But the bacterial flora evolves over time, and this evolution is often unfavorable in the reactions that are desired to intervene in the tanks. The problem of the drift of bacterial cultures is a general problem found, for example, in the pharmaceutical industry. In this industry, drift is avoided by operating in a sterile manner. But it is economically inconceivable to work in such conditions, for example, inside a factory for waste treatment. Culture devices such as those put into practice in the industry in the production of metabolites of commercial interest, or the biodegradation of waste or wastewater, for example, are confronted with the problem of contation by species that come from the external environment . The behavior of these crops under sterile conditions that imply the total confinement of the equipment, is a solution to the problem of contation by external species, but it is difficult to foresee for reasons of cost of treatment within applications such as the biodegradation of waste, or equally impossible to implement in extensive uses of microbial populations, such as in pond sludge treatment, for example. On the other hand, patent publication WO 00/34433 describes a technique that allows the selection and accelerated proliferation of live cells in suspension. By maintaining a constant cell concentration regime (turbidostat) under unlimited periods, the described device behaves as an automated selection procedure which at the same time elites the static variants of living cells, that is, living cells that stagnate in the ducts and in the containers, and privileges the dynamic variants that remain in suspension, which are increasingly adapted to the growing conditions. The industrialization of a device of this type in order to treat the volumes of more m3, even more tens or hundreds of m3, can be considered by simple homotyping, but its operation will be expensive for multiple reasons: - the means put into practice for the periodic transfer of one tank to another, as well as the periodic transfer of sterilization and rinsing fluids, and of crop additives, consume energy, - a significant consumption of sterilization and rinsing fluids, - the use of a single tank at the same time, the other is on hold, so it is useless for the procedure. However, it must possess all the necessary equipment for the development of the crop: temperature regulation, sterilization system, agitation system, etc. - the difficulty of continuously reading the turbidity within a large bioreactor vessel with important culture densities. For these reasons, the biodegradation of sewage so far applies different bacterial populations whose nature is beyond the control of the operator, and it is not possible to use only the species selected for their result or their effectiveness in relation to the substrate, that is, to the compounds that must be degraded, present in the wastewater. Therefore, it will be desirable to have a particular technique for biodegradation of wastewater, which makes it possible to essentially implement the selected species for their performance or for their effectiveness in relation to the compounds that must be degraded., present in the wastewater. Now, after long searches, the applicant society has discovered a procedure that allows reproducing and controlling proliferation conditions within a large container, or within a natural environment such as a pond or a pond, for example, and that works in a continuous, semi-continuous or discontinuous way, without resorting to confinement and sterilization, and privileging the dynamic variants of living cells, which are increasingly adapted to the growing conditions. This method is essentially based on cooperation between a bioreactor vessel and an automatic cell selection device. That is why the present application has as its object a method of continuous, semi-continuous or discontinuous treatment of a substrate, inside which said substrate installed inside a bioreactor vessel is subjected to the action of a culture of living cells C1 that allow effecting a reaction R1 on said substrate, and within which the medium is periodically and preferably regularly inoculated with the help of living C2 cells improving said reaction, said live C2 cells are the product of a selection made by an automatic selection device , preferably exclusively in suspension, of a population of living cells that are fed either to a different substrate or to the same substrate as the bioreactor vessel and are inoculated at the source by the living cells C1 present in the vessel of the bioreactor vessel for transferring them to the automatic selection device. If desired, other live cells can be added to the selection device or the bioreactor vessel at any time, for example to increase cell concentration or introduce novel species. In the present application, and in what follows, the term "dynamic living cells" designates living cells that proliferate in suspension, and which are subjected to a directed selection (in reverse of the "static living cells", which designates living cells). that adhere to the surface of the containers and conduits, thus escaping the selection). Static living cells are advantageously removed periodically. Generally, living cells that proliferate in suspension and that are subject to targeted selection will be used as living C2 cells. Within certain applications, dynamic living cells will not be used as live C2 cells, but live static cells. The substrate allows to maintain the cultures of living cells C1, C2, etc. Under preferred conditions of implementation of the invention, the device for automatic selection of dynamic living cells includes: - two or more containers that allow receiving and maintaining cultures of live cells in suspension, a set of means that allows these containers to be fed separately in sterilization, cleaning or neutralization fluids - a set of means that allows these containers to be supplied in gas - a set of means that allows these containers to be supplied in the substrate - a set of means that allows the content of one container to be transferred within the other and vice versa - a set of means that allows all or part of the content of these containers to be evacuated in another device such as a bioreactor vessel. - a set of means that allows to evacuate all or part of the contents of these containers in a garbage can. At the beginning of the implementation, the live C1 cells are present in the vessel of the bioreactor vessel, and in the automatic selection device, in the course of time, the selection device privileges (selects) the appearance and proliferation of variants of dynamic C2 living cells, always better adapted to the culture conditions and counter-selects the less well-adapted C1 living cells. Live C2 cells are transferred to the bioreactor vessel, or enter into competition with the living cells C1 and then impersonate them. In the end, it is found that the population of living cells C1 has been replaced by living C2 cells. Preferably, in parallel, living cells are retained in the vessel of the vessel for transfer into the automatic selection device. The automatic selection device of dynamic living cells comprises in particular (a) at least one first and at least one second culture vessel intended to receive a culture; (b) a gas source; (c) a source of medium; (d) a source for a sterilizing agent; and (e) a duct system including means for selectively connecting one of the two culture vessels to the medium source, such as valves, as well as the two culture vessels between them, and to selectively connect the other vessel. of culture with the source of the sterilizing agent. The case used can be adapted to live aerobic or anaerobic cells. In other preferred conditions of implementation of the invention, two connecting ducts including a common duct section are provided between the two culture vessels. In other preferred conditions of implementation of the invention, an evacuation conduit is provided on the common conduit section by means of which the cultures can be extracted from the culture vessels. Live C2 cells that improve the reaction, preferably are extracted by this conduit. In still other preferred conditions of implementation of the invention, - the bioreactor device and the automatic selection device are fed with the same substrate, - the bioreactor operates continuously, the production of substrate feed applied on the input line of substrate is identical to that applied in the transfer line of the culture medium. An automatic genetic selection device for live C2 cells that can be used is, in particular, that described in WO-A-00/34433, and that can operate in such culture conditions that the selection device always privileges the variant living cells, called " dynamics ", which are increasingly adapted to the culture conditions maintained within the bioreactor vessel. On the other hand, by periodically transferring the living cells of the bioreactor vessel in the automated selection device, it is certain to put the two living cell populations in competition and to select between possible variants that leave the bioreactor vessel and those of the automated selection device, living cells better adapted to the conditions of the bioreactor. The yields of the bioconversion or biodegradation process carried out in the bioreactor vessel are thus the worst maintained, but usually permanently improved thanks to the periodic substitution of the active living cells present in the bioreactor vessel by active living cells resulting from the automated selection device, increasingly powerful as they are always better adapted to the growing conditions. On the other hand, the inoculation is repeated periodically, and consequently the living cells are adapted better and better to the culture conditions, the automated selection device guarantees the preponderance of the most active living cells against those of the substrate present in the bioreactor vessel . A small automatic selection device, for example equipped with 25 mL culture vessels only, is sufficient to efficiently operate a bioreactor vessel such as the bio-ventilation tank of a 100 m3 wastewater treatment plant. It is possible, of course, to use culture vessels with a larger volume, for example, 1 liter. The C2 living cells used that improve the bioconversion reaction can be produced in particular by the implementation of a process that includes the following steps: (a) placing a crop in at least a first culture vessel; (b) continuous feeding of the culture into the first gas culture vessel, from a gas source and regular replenishment of liquids from a source of medium. (c) transferring the culture of the first culture vessel through the connecting conduits into at least a second culture vessel by means of an appropriate conduit circuit. (d) connecting the first culture vessel to a source with a sterilizing agent, to sterilize the first culture vessel. (e) extraction of the sterilizing agent from the first culture vessel, (f) continuous feeding of the culture into the second gas culture vessel from the gas source and regular replenishment of liquids from the medium source, (g) ) return of the culture of the second culture vessel through the connecting conduits, to the first culture vessel by means of a circuit with appropriate conduit, (h) connection of the second culture vessel to the source of the sterilizing agent, to sterilize the second vessel of culture; and (i) extraction of the sterilizing agent from the second culture vessel. In preferred conditions of implementation of the process described hereinabove, steps (b) to (h) are respected at least once. In the present application and in the following, the term "bioreactor vessel" designates, for example, the bio-ventilation tank of a purification station, the methanization tank of an anaerobic biological treatment unit, a pond, a pond, a tank, for example, from 0.5 liters to 100 m3, in particular from 1 liter to 100 m3, particularly from 5 liters to 50 m3 and more particularly from 10 liters to 50 m3, or a fermenter for example from 0.5 liters to 100 m3 , in particular from 1 liter to 100 m3, particularly from 5 liters to 50 m3, and more particularly from 10 liters to 50 m3. In the present application and in the following, the term "substrate" designates a medium containing a compound in which the metabolic conversion can be considered, in particular a water of industrial origin such as, for example, storage tank washing waters. hydrocarbons, washing water from production facilities of pharmaceutical intermediaries, rinsing water from filter cakes, flue gas from chemical products, effluents resulting from the melting of aircraft, water from municipal sources such as domestic wastewater, an accidental pollutant of the environment, such as the presence in the sea of a layer of hydrocarbons or other chemical products, respectively, from the sinking of an oil or chemical tanker, chemical effluents spilled on the ground after an accident involving an transport tanker (road or rail), soils contaminated with Heavy metals or with dioxins. The term "substrate" also designates a component in which the metabolic conversion is considered, for example, glucose, which serves for the production of biomolecules of industrial interest such as lysine, xanthan, alginates, polyols such as glycerol, hygromycin, ethanol which serves to the production of vinegar by acetic fermentation, oxalic acid used for applications of biohydrometallurgy, pectins and carrageenans. The term "substrate" still designates a medium containing living or dead cells where the metabolic conversion is considered, for example, an activated sludge from an urban or industrial wastewater, lignocellulosic derivatives produced by the paper industry, solid by-products or pasty products of the fishing industry, such as chitinous derivatives, for example, those produced by crabs or shrimp shells. The term "substrate" also designates contaminating molecules such as volatile organochlorine compounds (such as chlorinated solvents and CFCs), organochlorinated pesticides (such as DDT), halogenated polycyclic aromatic hydrocarbons (such as PCBs, dioxins and furans), solvents (such as benzene, toluene, xylene), organochlorine or organophosphorus phytosanitary compounds. The term "living cells" designates for example one or more bacterial flowers such as Sphingomonas wittichii (which catalyzes the bioconversion of cyanides and cyanates), Agrobacterium radiobacter (which catalyzes the bioconversion of pesticides such as bromoxynil), certain strains of Alcanivorax or Acinetobacter (able to biodegrade numerous aliphatic hydrocarbons), Xanthomonas campestris (which is involved in the biosynthesis of xanthano) or Sphingomonas paucimobilis (which is involved in the gelano biosynthesis). The term "living cells" also designates animal cells as mammalian cells (such as HEK-293 cells) for the production of monoclonal antibodies, of cellular factors for the development of insect cells for the production of recombinant proteins or of entomopathogenic viral particles. (like the Sf9 cells). The term "living cells" also refers to plant cells as plant cells of Datura for the production of tropic alkaloids (atropine, hyoscyamine and scopolamine), transgenic plant cells for the production of molecules of industrial interest (such as starch overproduction by potatoes). . The term "living cells" also designates algae as the green algae belonging to the Spyrogyra species involved in the biological treatment of effluents containing dyes such as Reagent Yellow 22, cultures of the micro-alga Scenedesmus quadricauda used for the bioconversion of the progesterone, Chlorella vulgaris micro-algae and Coenochloris pyrenoidosa that intervene in the biodegradation of p-chlorophenol, the macro-alga Microspora able to eliminate lead. The term "living cells" also designates yeasts such as Saccharomyces cerevisiae, which serve for the production of bioethanol from glucose or for the production of xylitol from glucose, Candida tropicalis and EC14 which serves for the biodegradation of phenolic compounds ( from the production of olive oil), Candida famata used for the biodegradation of nitrile compounds. The term "living cells" refers to fungi such as Penicillium janthinellum, capable of producing a zylanase, an enzyme that depolymerizes xylan, Streptomyces clavuligerus, capable of producing cephalosporin C from glucose as the sole source of carbon, or Phanerochaete chrysosporium, capable of biodegradation di- and tetrachlorinated dioxins. The term "living cells" also designates protozoa such as Euglena mutabilis (protozoan acidophilus) involved in the bioconversion of arsenic. The term "living cells" also means a mixture of all the types of living cells cited above. The periodic inoculation from the automatic cell selection device for example is carried out every 48 hours, preferably at least once a week, particularly at least once a month, and more particularly after each notable improvement in the rate of development of the cells. the living C2 cells. The methods of continuous, semi-continuous or discontinuous treatment of a substrate object of the present invention possess three interesting qualities. They make it possible in particular to biologically control the operation of a bioreactor of conventional design to exert control of the living cells present by eliminating living cells less adapted to the culture medium, as a contaminant having a growth rate lower than that of the cells live in the bioreactor vessel, for example. It is possible then to free yourself from the problems of sterility. A small selection device, for example, provided with one liter bioreactor vessels, is sufficient to efficiently operate a vessel such as a water tank with a volume of 4000 m3. The invention also makes it possible to improve the efficiency of a culture method of conventional design, by increasing the activity of the living cells used in the process without remelting the devices used. The production yields of a molecule of interest and / or the degradation rate of substrates can also be increased. These qualities are illustrated here below, in the experimental part. They justify the use of the procedures described here above, for example in the biodegradation of recalcitrant compounds. In fact, today a large amount of waste emitted by the chemical industry is destroyed by incineration with high costs, and with a significant environmental risk linked to the risk of emissions to the atmosphere of dangerous compounds for humans and their environment. Biological treatment of these wastes (or bioconversion) is often made impossible by the necessary treatment times, or by the inability of living cells to metabolize the compounds present in the waste, or even by the inhibitory effect of certain compounds against the waste. the bacterial activity in general. The device of the invention makes it possible to maintain, within a bioreactor vessel dedicated to the bioconversion of waste, specifically adapted living cells, competent against the compounds present in the waste and then make possible the bioconversion of waste traditionally destroyed by incineration. By selecting, thanks to the selection device, the living cells increasingly adapted to the culture medium, the invention makes it possible to improve the efficiency of a conventional culture method by increasing the activity of the living cells used in the process without remelting the put into practice. These qualities also justify the use of the methods described herein, for example, in the improvement of the operation of the stations for the biological treatment of effluents. In fact, the proper functioning of the purification stations of urban or industrial effluents can be affected by the accidental presence in the effluents of recalcitrant compounds against the living cells present. It can be envisaged to remedy this problem by attachment of a device such as that described hereinafter. The bioreactor vessel in this case is materialized by the bio-ventilation tank in the purification station. The feeding of the automated selection device can be performed by a chopping previously located in the ventilation system in a primary decanting vessel for example. The reciprocal inoculation of the selection device and the bioreactor vessel is carried out as illustrated hereinafter in FIG. 1. An external connection line can also be used to feed the robot with a modified substrate in relation to the medium previously taken from the tank. of bio-ventilation. This device can be used to enrich the bio-ventilation deposits in living cells adapted to the degradation of possible recalcitrant compounds present in the effluents. These qualities also justify the use of the methods described hereinafter, for example, in the improvement of biosynthesis yields. The invention can be used for a certain effect, for the improvement of performance (yield, development time) of industrial synthesis processes by biocatalysis. Today, the improvement of the performance of biosynthesis resting on fermentation, either in batches or continuous, is done by optimizing the composition of the culture medium and physical-chemical parameters of the crop (temperature, oxygenation, pH, etc.). ). These developments are long and expensive and in any case are limited by the metabolism of the living cells that are present. The invention, by acting on the metabolism of the living cells present, makes it possible to adapt said living cells to the conditions imposed by the technical-economic imperatives and to increase the rate of growth, and consequently consequently to increase the overall results of the biosynthesis. In the case, for example, of the yeasts, said osmotolerants are involved in the production of polyols (sorbitol), mannitol, xylitol, etc.), variable culture times of 4 to 5 days are observed on glucose concentrations of approximately 30 g / L. It is possible, thanks to the invention, to bring the yeasts into contact with increasingly important concentrations of glucose, in order to orient their natural metabolism towards the production of the desired extracellular or intracellular metabolite, after increasing their rate of growth, that is to say when decreasing the times needed for cultivation. This improvement translates into an increase in the productivity of the equipment used, and a decrease in the cost of biosynthesis. The method according to the invention can be implemented over long periods of time, as well as indefinitely. The subject of the present application is a live cell culture device comprising: A: a selection device, preferably including two or more containers that allow receiving and maintaining cultures of live cells in suspension, a set of means that allow these containers to be supplied separately in sterilization, cleaning or neutralization fluids - a set of means for supplying these containers in gas - a set of means for supplying these containers in the substrate - a set of means for transferring the content of the containers. one container inside the other and vice versa - optionally, a set of means that allow to evacuate all or part of the content of these containers in another device such as a bioreactor container. - a set of means that allow to evacuate all or part of the contents of these containers in a garbage can. - B: a bioreactor vessel, - C: a system of means for transferring live cells between the selection device and the bioreactor vessel, - D: optionally, a conduit composed of means for attaching the bioreactor vessel to a solid separation device. liquid such as a decanter, - E: optionally, a conduit for evacuation of fluid (water, for example), treated - F: optionally, a temperature regulation device. The means for transferring the contents of one container in the other and vice versa, can be physical means, such as conduits, or human means that effect the extraction of one to transfer it in the other. The present application, more particularly, has as its object a live cell culture device by coupling with a live cell selection automaton, comprising: A: a live cell selection device, comprising (a) at least one first and at least one second culture vessel intended to receive a culture (b) a gas source (c) a source of medium (d) a source for a sterilizing agent; and (e) a duct system constituted by means for connecting, optionally, one of the two culture vessels with the medium source, such as valves, as well as the two culture vessels between them, and to selectively connect the another culture vessel with the source of the sterilizing agent. - B: a bioreactor vessel. - C: a system of means for transferring live cells between the selection device and the bioreactor vessel, - D: optionally, a conduit including means for connecting the bioreactor vessel with a solid-liquid separation device, such as a decanter . - E: optionally, a fluid evacuation conduit (water for example), treated. - F: optionally, a temperature regulation device. Under preferred conditions of implementation of the invention: a feed line is installed between the bioreactor vessel and the automatic selection device to allow the extraction of living cells present in the bioreactor vessel in order to make them evolve in the automatic selection device , - an inoculation line is installed between the automatic selection device and the bioreactor vessel to allow the bioreactor vessel to be repeatedly and regularly inoculated with living cells that have evolved in the automatic selection device. - a supplementary line allows enriching the culture medium of the automaton with one or more additives, - a collection tank for rinsing and sterilization effluents allows to collect the sterilization and rinsing fluids of the automatic selection device, - a set of pumps allows the transfer of different fluids. The preferred conditions of implementation of the methods described hereinabove apply equally to the other objects of the invention mentioned above, in particular to the devices for their implementation. The invention will be better understood if reference is made to the accompanying drawings, in which - Figure 1 represents a schematic view of a device of the invention, - Figure 2 represents a schematic view of a biological sewage treatment device, - Figure 3 represents a schematic view of an automatic selection device described in WO 00/34433. On FIG. 1, a bioreactor container 1 connected by a system of return ducts 5 and flow 6 of an automatic crop selection device 2 can be observed. Pumps 13, 14 are provided on these ducts. It is also possible to observe a buffer tank 11 of an externally supplied substrate connected by a duct system 4 on the one hand to the automatic crop selection device 2, and on the other hand to the bioreactor container 1. Pumps 9, 10 are provided on these conduits. A tank 8 for collecting rinsing and sterilizing effluents from the automatic live cell selection device 2 is provided. An additive input conduit 12 is provided for the addition in the automated live cell selection device 2. The device can work in particular as follows: The bioreactor vessel 1 and the automatic selection device 2 are fed with the same substrate respectively through tracks 3 and 4. Bioreactor 1 operates continuously, the production of substrate feed applied on line 3 is identical to that applied on line 7 corresponding to the transfer of culture medium. Line 7 can lead to a solid-liquid separation device, not shown, such as a decanter. An inoculation line 5 installed between the automatic selection device 2 and the bioreactor vessel 1 makes it possible to inoculate the bioreactor vessel 1 repeatedly and regularly with living cells that have evolved within the automatic selection device 2. A transfer line 6 installed between the bioreactor vessel 1 and the automatic selection device 2 allows live cells present in the bioreactor vessel 1 to be taken in order to make them evolve in the automatic selection device 2.

A supplementary line 12 makes it possible to enrich the culture medium of the automaton with one or more additives. A rubbish bin 8 allows to collect the sterilization and rinsing fluids of the automatic selection device. A set of pumps 9, 10, 13 and 14 allows the transfer of the different fluids. On figure 2, a biological sewage treatment device can be observed. A part of the above elements can be observed, namely, a bioreactor vessel 1 which is an aeration tank and an automated selection device 2, fed with the same substrate respectively through tracks 3 and 4, some inoculation lines and of transfer 5 and 6 installed between the bioreactor vessel 1 and the automated selection device 2, and a conduit 15 including means for connecting the bioreactor vessel 1 with a solid-liquid separation device, in this case, a decanter 16. Figure 3, a first and a second culture vessel 20, 21, intended to receive a culture 22, a gas source 23, a source of medium 24, a source 25 for a sterilizing agent, and a system of ducts including means for connecting, optionally, one of the two culture vessels 20 or 21 with the source 25 of the sterilizing agent. The lines in thick line represent the active conduits in the case of one of the phases of implementation of the procedure.

This device allows the provision of a crop 22 in at least one first culture vessel 20, the continuous feeding of the culture 22 in the first culture vessel. 20 with gas from a gas source 23 and regular replenishment in liquids from a source of medium 24, the transfer of the crop 22 from the first culture vessel 20 by connecting conduits 28-31 into at least a second container of culture 21 in the middle of an appropriate conduit circuit, the connection of the first culture vessel 20 with a source 25 by a sterilizing agent, to sterilize the first culture vessel 20, the removal of the sterilizing agent from the first culture vessel 20, the continuous feeding of the culture 22 in the second culture vessel 21 with gas from the gas source 23 and regular replenishment in liquids from the source of medium 24, the return of the culture 22 of the second culture vessel 21 by the connecting conduits 28-31 in the first culture vessel 20 in the middle of an appropriate conduit circuit, the connection of the second culture vessel 21 with the source 25 for the sterilizing agent, to sterilize the second culture vessel 21. The following examples illustrate the present application.

EXAMPLE 1: BIOCONVERSION OF WASTE RESULTING FROM THE PRODUCTION OF PESTICIDE PRODUCTS It is continuously fed at a fixed rate of 0.75 mL / min, a bioreactor vessel of 5 useful liters, with a substrate containing waste resulting from the production of pesticide products. The analysis of these wastes reveals the following chemical compounds: alcohols (example: 2-butoxyethanol), allies (example: 2-2-dimethoxypropane), chlorophenols (example: 2,4-dichlorophenol), aromatics (example: 1, 1 '-biphenyl, 1-methylnaphthalene, 2-methylnaphthalene, 2-ethylnaphthalene), brominated compounds (example: 3,3-dibromo-4-hydroxybenzonitrile) and pesticides (example: 2,4-D-butoxyethyl ester, CP and CPP). The chemical oxygen demand of this waste is 2450 mg / L. The bioreactor vessel is inoculated with 10 mL of a mixture of live cells of microorganisms isolated from intakes from different ecological niches or activated sludge from purification stations; These living cells are selected because they are able to degrade waste. The continuous regime is maintained by setting a residual DCO of 1000 mg / L, which represents a stabilized bioconversion yield of 59.18%. On the other hand, an automatic selection device of the type described in FIG. 1 of WO-A-00/34433 provided with 25 ml culture vessels with the same substrate and inoculated with the same mixture of living cells as previously was also fed. . At the beginning, inside the two vessels the same living cells are then present.

As soon as the turbidity target is reached for the selection system (turbidostat detection), the bioreactor vessel is inoculated automatically with 10 mL of the medium present in the automatic selection device. After 9 weeks of operation, it is verified that the live cells that leave the automatic selection device, where the growth rate has doubled during this period (going from 0.009 to 0.018 n "), have replaced the population of living cells present At the origin in the bioreactor vessel, in this final state, only two microorganisms could be identified as Delftia acidovorans and Pseudomonas putida A. Therefore, it was possible to increase the fermenter's power production by 100% while maintaining the same bioconversion yield.

Claims (13)

- 28 - CLAIMS

1. A process for continuous, semi-continuous or discontinuous treatment of a substrate (24) installed in a bioreactor vessel (1), said substrate is subjected to the action of living cells C1 that allow to carry out a reaction R1, characterized in that it comprises the following steps: a) living C1 cells are taken from the substrate present in the vessel of the bioreactor vessel (1) to transfer them into an automatic selection device (2) of a population of living cells that proliferate in suspension, said device being fed by a different substrate, either by the same substrate (24) as the bioreactor vessel (1), in order to obtain living C2 cells that allow increasing the yield of the reaction R1, and b) said substrate is periodically inoculated with the help of novel ones live C2 cells obtained in the preceding stage.

2. A method according to claim 1, further characterized in that the automatic selection device (2) of the cells that proliferate in suspension comprises: - two (20, 21) or more containers, which allow to receive and maintain cell cultures live in suspension. - a set of means for separately feeding these containers in sterilization (25), cleaning or neutralization fluids, - a set of means for feeding these gas receptors (23), - a set of means which allows feeding these containers on substrate (24), - a set of means (28-31) that allows transferring the contents of one container (20) in the other (21) and vice versa, - a set of means that allows to evacuate all or part of the contents of these containers in another device, such as a bioreactor vessel (1). - a set of means that allows all or part of the content of these containers (20, 21) to be evacuated to a garbage can.

3. A method according to claim 1 or 2, further characterized in that the device for automatic selection of the cells that proliferate in suspension comprises in particular: - at least one first and at least one second culture recipient (20, 21) intended to receive a culture (22), - a gas source (23), - a source of medium (substrate) (24) - a source (25) for a sterilizing agent, and - a system of conduits comprising means for selectively connect one of the two culture vessels (20 or 21) with the medium source (24), such as valves, as well as the two culture vessels (20, 21) between them, and to selectively connect the another culture vessel (20 or 21) to the source (25) of the sterilizing agent. - 30 -

4. A method according to one of claims 1 to 3, further characterized in that the live C2 cells are a product of the selection made between a population of living cells that proliferate exclusively in suspension. A method according to one of claims 1 to 4, further characterized in that the bioreactor vessel (1) is a bio-ventilation tank of a purification station, the methanization tank of an anaerobic biological treatment unit, a lagoon, a pond of water, a tank from 0.5 liters up to 100 m3, or a fermenter. 6. A process according to one of claims 1 to 5, further characterized in that the C2 living cells used that increase the yield of the bioconversion reaction can be produced in particular by the implementation of a process comprising the following steps: (a) making available a crop (22) in at least one first culture vessel (20), (b) continuously feeding the culture (22) into the first culture vessel (20) with gas from a source of gas (23) and regular replenishment in liquids from a substrate source (24), (c) transfer of the crop (22) from the first culture vessel (20) by connecting conduits (28-31) within minus a second culture vessel (21) in the middle of an appropriate conduit circuit, - 31 - (d) connecting the first culture vessel (20) with a source (25) for a sterilizing agent, for sterilizing the first culture vessel (20), (e) removing the sterilizing agent from the first culture vessel (20), (f) continuous feeding of the culture (22) into the second culture vessel (21) with gas from the gas source (23) and regular replenishment in liquids from the medium source (24), (g) returning the culture (22) of the second culture vessel (21) through the connecting conduits (28-31) into the first culture vessel (20) in the middle of an appropriate conduit circuit. (h) connecting the second culture vessel (21) with the source (25) for the sterilizing agent, for sterilizing the second culture vessel (21), and (i) removing the sterilizing agent from the second culture vessel (21) . A process according to one of claims 1 to 6, further characterized in that the substrate (24) is - a medium containing a compound where metabolic conversion is foreseen, for example, a water of industrial origin, a water of municipal origin eg domestic sewage, an accidental pollutant from the environment, for example, the presence in the sea of a layer of hydrocarbons or other chemical products, a chemical effluent spilled on the ground, a soil contaminated with metals heavy or with dioxin, or - a compound where the metabolic conversion is foreseen, for example, of glucose, ethanol, or oxalic acid, or - a volatile organochlorine compound, an organochlorine pesticide, a halogenated polycyclic aromatic hydrocarbon or a solvent. 8. A method according to one of claims 1 to 7, further characterized in that the living cell comprises one or more bacterial species, animal or plant cells, algae, yeast, or fungi. 9. A method according to one of claims 1 to 8, further characterized in that the periodic inoculation from the automatic selection device (2) of living cells is carried out at least once a week. 10. A live cell culture device, further characterized in that it comprises: A: a bioreactor vessel (1) capable of containing living cells C1 that perform a R1 reaction of bioconversion of a substrate (24); - B: a live cell selection device that privileges the selection of live C2 cells that proliferate in suspension, said live C2 cells are variants that are derived from live C1 cells initially presented in the bioreactor (1), and said C2 cells allow to increase the yield of the R1 reaction of bioconversion of said substrate (24); - C: a system of conduits (5) comprising means for operating transfers of the selection device (2) to the bioreactor vessel (1); and - D: a duct system (6) comprising means for operating transfers from the bioreactor vessel (1) to the selection device (2); - E: optionally a conduit (15) comprising means for connecting the bioreactor vessel (1) with a solid-liquid separation device such as a decanter (16); - F: optionally, a conduit for evacuation of fluid (water, for example) treated; - G: optionally, a temperature regulation device. A device for the cultivation of living cells according to claim 10, further characterized in that the selection device (2) comprises: - two containers (20, 21) or more, which allow receiving and maintaining cultures of living cells in suspension; - a set of means for feeding these containers on substrate (24); - a set of means (28-31) that allow transferring the contents of one container (20) in the other (21) and vice versa; - a set of means that allow all or part of the content of these containers to be evacuated to another device, such as a bioreactor vessel (1); - a set of means that allow all or part of the content of these containers (20, 21) to be evacuated to a garbage can. 12. A live cell culture device according to claims 10 or 11, further characterized in that the device for selecting living cells that proliferate in suspension comprises: - at least one first and at least one second culture vessel (20). , 21) intended to receive a culture (22) - a gas source (23) - a source of medium (2) - a source (25) for a sterilizing agent; and - a duct system comprising means for selectively connecting one of the two culture vessels (20 or 21) to the source of medium (24), such as valves, as well as the two culture vessels (20, 21) between them, and to connect optionally the other culture vessel (20 or 21) to the source (25) of the sterilizing agent. 13. A device according to any of claims 10 to 12, further characterized in that the bioreactor vessel (1) is a bio-ventilation tank of a purification station, the methanation reservoir of a biological treatment unit. anaerobic, a lagoon, a water tank or a tank from 0.5 liters to 100 m3.